Replication of the HIV-1 genome, like all nucleic acids, involves synthesis of a minus and a plus strand. Despite the application of quantitative real-time PCR technology, several questions regarding the complex nature of reverse transcription remain unanswered, including the rates of RNA- and DNA-dependent DNA synthesis during infection. Currently available PCR methods cannot distinguish between the two strands. Because reverse transcription occurs on both strands simultaneously, and has the potential to copy the same minus strand multiple times through displacement synthesis, it is not possible to analyze the kinetics of reverse transcription using conventional PCR. To determine the rates of HIV-1 reverse transcription in infected cells, we have developed a novel SSA assay using single-stranded padlock probes that are specifically hybridized to either the minus strand or the plus strand, ligated, and quantified using real-time PCR. Using SSA, we have determined for the first time the rates of HIV-1 RNA-dependent DNA synthesis in 293T and human primary CD4+ T cells. We will use a strand-specific quantitative real-time PCR assay previously developed in our lab to gain insights into the mechanisms of reverse transcription in cell culture. We will characterize the effects of NRTI and NNRTI resistance mutations on the kinetics of various steps in reverse transcription. We will analyze the kinetics of reverse transcription in macrophages using strand-specific amplification to determine whether lower dNTP pools result in slower reverse transcription. We will develop a cell culture assay for viral uncoating and determine the kinetics of uncoating, the intracellular location of uncoating, and the relationship between viral uncoating and the kinetics of reverse transcription. [Corresponds to Pathak Project 3 in the April 2007 site visit report of the HIV Drug Resistance Program]